The objectives of this research program are to investigate the purified alpha and alpha beta DNA polymerases and their associated ribonuclease H (RNase H) activity found in avian myeloblastosis virus (AMV). This system is unique for studying because no system has been developed where one can compare two structurally different DNA polymerases which originate from one species of RNA tumor virus. This study will be directed toward the following goals: A) To investigate the mode of action of RNase H activity associated with the alpha AMV DNA polymerase. The alpha beta RNase H has been shown to be a processive exonuclease. Several other RNA tumor viruses purified DNA polymerase-RNase H complexes will be studied to compare the RNase H mechanism of avian, murine, and feline viruses. The mechanism of RNase H nuclease activity will be investigated by using gel electrophoresis to investigate product size of digested substrates, using covalently closed RNA of RNA-DNA hybrids to determine if free ends are needed for initiation of enzymatic activity, and P32 end labled H3 poly(A) to measure the release of the end label with respect to the entire molecule. Alpha beta and E. coli (an endonuclease) RNase H will be utilized as controls. B) To determine the extent of transcription, the size, and the sequence complexity of the viral specific DNA synthesized by alpha and alpha beta AMV polymerase using different RNA tumor virus 70S RNAs, and, the DNA products made by alpha and alpha beta polymerase using rabbit globin mRNA with oligo(dt) present will also be investigated. The above systems will be evaluated by hybridization studies and reassociation kinetics (Cot and Crt). C) To establish the relationship of alpha and alpha of alpha beta by using peptide fingerprint mapping and N and C-terminal amino acid analysis. D) To select appropriate proteolytic conditions to separate the DNA polymerase and RNase H active sites on alpha and alpha beta DNA polymerase followed by evaluation of these separated activities.